Although prostaglandin E2 (PGE2) is known to inhibit glucose-induced insulin secretion, it is uncertain whether PGE2 actions on the β-cell are direct, whether they are equipotent for both phases of hormone secretion, and whether the same mechanism of action prevails throughout. Study of the HIT cell, a clonal line of pancreatic β-cells, provides answers to these questions because perifusion with glucose and 3-isobutyl-1-methylxanthine stimulates biphasic insulin secretion. Perifusion with PGE2 decreased both the first and second phases of glucose-induced insulin release to 47 ± 4% of controls. Pretreatment with pertussis toxin partly prevented PGE2 inhibition to 80 ± 4% of controls for first phase and 79 ± 4% of controls for second phase. To evaluate whether the partial prevention of PGE2 inhibition seen with pertussis toxin pretreatment was caused by G(i) heterotrimer association between the preincubation period and the end of perifusion, PGE2 actions were also examined during continuous treatment with pertussis toxin. Under these conditions, PGE2 inhibition of both phases was totally prevented. However, no difference was observed in membrane protein ADP ribosylation when cells were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after pretreatment or continuous treatment with pertussis toxin. Cyclic AMP (cAMP) accumulation was inhibited by PGE2 (from 3263 ± 153 to 1549 ± 158 fmol/106 cells) but less so after pretreatment with pertussis toxin (correlation between insulin release and cAMP accumulation during perifusion; n = 18, r = .85, P < .001). Thus, PGE2 equally inhibits both phases of glucose-induced insulin secretion and cAMP generation through a pertussis toxin-sensitive G protein-mediated direct effect on the pancreatic β-cell.